The tocopherols and plastochromanol-8 (PC-8) content were determined according to Siger et al. [46 (link)]. Briefly, oil was dissolved in n-hexane and transferred to vials for analyses. The tocopherol and plastochromanol-8 content were analysed using a Waters HPLC system (Waters, Milford, MA, USA) equipped with a LiChrosorb Si 60 column (250 × 4.6 mm, 5 µm, Merck, Darmstadt, Germany), a fluorimetric detector (Waters 474) and a photodiode array detector (Waters 2998 PDA). The mobile phase was a mixture of n-hexane with 1.4-dioxane (96:4, v:v). The flow rate was 1.0 mL/min (for tocopherols and PC-8). To detect the fluorescence of tocopherols and PC-8, the excitation wavelength was set at ʎ = 295 nm and the emission wavelength at ʎ = 330 nm. Standards of α-, β-, γ- and δ-tocopherols (>95% of purity) were purchased from Merck. The tocopherol standards were dissolved in n-hexane to obtain ten different concentration levels, in the range from 1 to 20 µg/mL, and injected to the HPLC system at 10 µL. The determination coefficient, which expresses the linearity of the standard curve, is equal to 0.99 and it is statistically significant (p < 0.0001) in case of all investigated standards. The limits of detection (LODs) for T were as follows: 0.86, 0.57, 0.64, 0.82, 0.89, 0.93, 0.75, 1.41, and 0.75 µg/mL for α-T, β-T, γ-T and δ-T respectively.
Lichrosorb si 60 column
Manufactured by Merck Group
Sourced in Germany, United States
LiChrosorb Si 60 column is a chromatographic column used for the separation and purification of various organic compounds. It is packed with a silica-based adsorbent material, providing a stationary phase for the separation process. The column is designed to facilitate efficient separation and analysis of compounds based on their interactions with the stationary phase.
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Lab products found in correlation
Waters hplc system, by Waters Corporation (5 mentions)
Hplc system, by Waters Corporation (3 mentions)
Jetstream 2 plus, by Merck Group (3 mentions)
E2695 hplc system, by Waters Corporation (1 mentions)
Hplc fd, by Shimadzu (1 mentions)
F254 plate, by Merck Group (1 mentions)
Mercury plus 400 nmr spectrometer, by Agilent Technologies (1 mentions)
Sx102a mass spectrometer, by JEOL (1 mentions)
Waters jetstream 2 plus, by Waters Corporation (1 mentions)
16 protocols using lichrosorb si 60 column
1
Tocopherol and Plastochromanol-8 Quantification
2
Quantifying Tocopherol Content in Oils
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The tocopherols contents were determined according to Siger et al. [32 (link)]. Briefly, oil was dissolved in hexane and transferred to vials for analyses. The tocopherols contents were analyzed by using Waters HPLC system (Waters, Milford, MA) equipped with a LiChrosorb Si 60 column (Merck, Darmstadt, Germany) (250 mm, 4.6 mm, and 5 µm) and a fluorimetric detector (Waters 474) and a photodiode array detector (Waters 2998 PDA). The mobile phase was a mixture of hexane with 1,4-dioxane (96:4 v/v). The flow rate was 1.0 mL/min (for tocopherols and PC-8). To detect the fluorescence of tocopherols, the excitation wavelength was set at ʎ = 295 nm, and the emission wavelength was set at ʎ = 330 nm.
3
Tocochromanol Content Analysis in Oils
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The total tocochromanol content and composition were determined according to Siger et al. [49 (link)]. The oil was dissolved in n-hexane and transferred to vials for analysis. The tocochromanol content was analyzed using Waters HPLC system (Waters, Milford, MA, USA) equipped with a LiChrosorb Si 60 column (Merck, Darmstadt, Germany) (250 mm, 4.6 mm, 5 μm), a fluorimetric detector (Waters 474), and a photodiode array detector (Waters 2998 PDA). The mobile phase was a mixture of n-hexane with 1.4-dioxane (96:4 v/v). The flow rate was 1.0 mL/min (for tocochromanols). To detect the fluorescence of tocochromanols, the excitation wavelength was set at ʎ = 295 nm and the emission wavelength at ʎ = 330 nm. Standards of all tocochromanols (>95% of purity) were purchased from Merck (Darmstadt, Germany).
4
HPLC Analysis of Tocopherols in Coffee Oil
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Tocopherols were determined according to the Chinese National Standard Method (GB/T 26635-2011). Briefly, green coffee oil samples (0.5 g) were diluted in 2.0 mL of n-hexane and filtered through a 0.22 µm nylon filter before HPLC analysis. Chromatographic separation was carried out on a Waters e2695 HPLC system (Waters Corp., Milford, MA, USA) equipped with a 3300 evaporative light scattering detector (ELSD; Grace Inc., USA), and separation was carried out by a Lichrosorb Si 60 column (250 mm × 4.6 mm, 5 µm, Merck, Darmstadt, Germany). To detect the fluorescence of tocopherols, the excitation wavelength was set at 295 nm, the emission wavelength was set at 330 nm, the injection volume was 10 µL, the mobile phases were composed of methanol:water (v/v = 98:2), and an isocratic elution programme was used at a flow rate of 1.0 mL/min. Tocopherols were quantified by establishing a standard curve with α-tocopherol, δ-tocopherol, β-tocopherol, and γ-tocopherol standard solutions.
5
Profiling Tocopherol Composition in Oils
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For the determination of α-, γ-, and δ-tocopherols, ~200 mg of oil sample was diluted in 1.8 mL of n-hexane, filtered through a 0.20 μm PTFE syringe filter, and analyzed by a high-performance liquid chromatography system coupled to fluorescence detector (HPLC-FD, Shimadzu, Milan, Italy) according to the conditions already reported by Albergamo and coworkers [35 (link)]. Specifically, the chromatographic separation was carried out by a LiChrosorb® Si60 column (250 mm × 4.6 mm I.D., 5 µm particle size, Merck, Darmstadt, Germany), protected by a LiChroCART 4-4 guard column with the same stationary phase (Merck, Darmstadt, Germany), and by exploiting a mobile phase consisting of n-hexane/ethyl acetate (90:10 v/v) under isocratic conditions. HPLC-FD analyses were performed at 40 °C with an injection volume of 20 µL and a flow rate of 0.8 mL/min. Data processing occurred by the LabSolutions software, ver. 5.10.153 (Shimadzu). The identification of tocopherols was carried out by a direct comparison with the retention time of relative commercial standards at respective excitation and emission wavelengths of 295 nm and 330 nm. The quantitative analysis was performed by constructing appropriate external calibration curves for every investigated tocopherol.
6
Nuclear Magnetic Resonance and Mass Spectrometry Analysis
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NMR spectra were analyzed in CDCl3 at room temperature using a Varian Mercury plus 400 NMR spectrometer, and the solvent resonance was used as the internal shift reference (tetramethyl silane [TMS] as standard). EI–MS were recorded on a SX-102A mass spectrometer (JEOL USA, Inc., Peabody, MA, USA). Thin-layer chromatography was used on silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany), and the spots were visualized by spraying with 10% H2SO4 solution. Silica gels (230–400 mesh ASTM, Merck KGaA) were used for column chromatography. Semi-preparative HPLC was performed using LiChrosorb Si 60 column, (7 μm, 250 × 10 mm; (Merck KGaA) on a LDC Analytical-III system.
7
Tocopherol Content Analysis by HPLC
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The tocopherols content was determined according to Siger et al. [61 (link)]. The tocopherols content was analyzed using a Waters HPLC system (Waters, Milford, MA, USA) equipped with a photodiode array detector (Waters 2998 PDA), a fluorimetric detector (Waters 474), and a LiChrosorb Si 60 column (250 × 4.6 mm, 5 µm, Merck, Darmstadt, Germany). The HPLC conditions were as follows: mobile phase was a mixture of n-hexane with 1.4-dioxane (96:4, v:v); the flow rate was 1.0 mL/min; injection sample volume was 10 mL; the excitation wavelength was set at λ = 295 nm and the emission wavelength at ʎ = 330 nm. The tocopherols were identified by comparison with retention times of standards purchased from Merck (>95% of purity).
8
Analysis of Tocopherols by HPLC
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The qualitative and quantitative determination of tocopherols were carried out using a Waters HPLC system (Waters, Milford, MA, USA), consisting of a pump (Waters 600), a fluorimetric detector (Waters 474), a photodiode array detector (Waters 2998 PDA), an autosampler (Waters 2707), a column oven (Waters Jetstream 2 Plus), and a LiChrosorb Si 60 column (250 × 4.6 mm × 5 µm) from Merck (Darmstadt, Germany). A mixture of n-hexane and 1,4-dioxane (96:4, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. The fluorescence of tocochromanols was detected at an excitation wavelength of 295 nm and an emission wavelength of 330 nm [53 (link)].
9
Tocochromanol Quantification in Plant Samples
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The tocochromanols (tocopherols, tocotrienols, and plastochromanol-8 (PC-8)) content was determined according to Siger et al. [40 (link)]. The tocochromanols content was analyzed using a Waters HPLC system (Waters, Milford, MA, USA) equipped with a LiChrosorb Si 60 column (250 × 4.6 mm, 5 μm, Merck, Darmstadt, Germany), a fluorimetric detector (Waters 474), and a photodiode array detector (Waters 2998 PDA). The conditions during the analysis were as follows: mobile phase was a mixture of n-hexane with 1.4-dioxane (96:4, v:v); the flow rate was 1.0 mL/min; injection sample volume was 10 mL; the excitation wavelength was set at ʎ = 295 nm and the emission wavelength at ʎ = 330 nm. The tocochromanols were identified by comparison with retention times of standards purchased from Merck (>95% of purity).
10
Tocopherol Analysis by HPLC
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Tocopherol content was determined following Gawrysiak-Witulska et al. [20 (link)]. HPLC was used for the determination of tocopherols and the method is described in detail in Hassanein et al. [21 (link)]. A LiChrosorb Si60 column (250 × 4.6 mm; 5 µm; Merck KGaA, Darmstadt, Germany) was used.
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